The use of optically detectable labeling groups, and particularly those groups having high quantum yields, e.g., fluorescent or chemiluminescent groups, is ubiquitous throughout the fields of analytical chemistry, biochemistry and biology. In particular, by associating a highly visible signal with a given reaction, one can better monitor that reaction as well as any potential effectors of that reaction. Such analyses are basic tools of life science research in genomics, diagnostics, pharmaceutical research, and related fields.
To date, such analyses have generally been performed under conditions where the amounts of reactants are present far in excess to compensate for any damage caused by the detection system and allow for signal detection with minimal impact on the reactants. For example, analyses based upon fluorescent labeling groups generally require the use of an excitation radiation source directed at the reaction mixture, to excite the fluorescent labeling group, which is then separately detectable. However, one drawback to the use of optically detectable labeling groups is that prolonged exposure of chemical and biochemical reactants to such light sources, alone, or when in the presence of other components, e.g., the fluorescent groups, can damage such reactants. The traditional solution to this drawback is to have the reactants present so far in excess that the number of undamaged reactant molecules outnumbers the damaged reactant molecules, thus minimizing the effects of the photodamage.
A variety of analytical techniques currently being explored deviate from traditional conditions. In particular, many reactions are based upon increasingly smaller amounts of reagents, e.g., in microfluidic or nanofluidic reaction vessels or channels, or in “single molecule” analyses. Such low reactant volumes are increasingly important in many high throughput applications, such as microarrays.
The use of smaller reactant volumes offers challenges to the use of optical detection systems. When smaller reactant volumes are used, damage to reactants, such as from exposure to light sources for fluorescent detection, can become problematic and have a dramatic impact on the operation of a given analysis. This can be particularly detrimental, for example, in real time analyses of reactions that include fluorescent reagents that can expose multiple different reactant components to optical energy. In addition, smaller reactant volumes can lead to limitations in the amount of signal generated upon application of optical energy.
As such, the present invention is directed to methods and compositions that result in increased effective concentrations of reactants and detection molecules in smaller reactant volumes, resulting in an increased signal within the smaller volume. In particular, the present invention provides methods and compositions to prevent or mitigate the adverse effects of photodamage in such reactions.